Embodiments of the invention relate generally to the detection, enumeration, and identification of replicating cells, especially microbial cells, and, more particularly, to a flat panel imaging system for acquiring digital images of microbial samples.
Microbial culture is the predominant methodology for the detection, enumeration, and identification of replicating cells, especially microbial cells (e.g., bacteria, yeasts, and molds), in medical, industrial, and environmental samples. Microbial culture provides for the detection of small numbers of microbes, as it allows for the simple visual detection of microbes by exploiting their propensity to reproduce in large numbers rapidly. A related microbial culture technique, called microbial enumeration or colony counting, quantifies the number of microbial cells in a sample. The microbial enumeration method, which is based on in situ microbial replication, generally yields one visually detectable “colony” for each microbial cell in the sample. Thus, counting the visible colonies—either manually by eye or thru an electronic/automated method—allows microbiologists to determine the number of microbial cells in a sample accurately. To perform microbial enumeration, bacterial cells can be dispersed on the surface of nutrient agar in petri dishes (“agar plates”) and incubated under conditions that permit in situ bacterial replication. The individual, visually undetectable, microbe replicates repeatedly to create a large number of identical daughter microbes at the physical site where the progenitor microbial cell was deposited. The daughter cells remain co-localized (essentially contiguous) with the original cell, so that the cohort of daughter cells (which may grow to tens or hundreds of millions of cells) eventually form a visible colony on the plate.
In addition to quantifying the number of microbial cells in a sample via microbial enumeration or colony counting, it is often desired to perform additional analysis on microbial cells in the sample. For example, potential treatments and administering of medicine (i.e., antibiotics) to the cells may often be performed to analyze the effectiveness of the medicine in treating the cells. Such analysis is often performed in conjunction with a technique that allow for image capture of the sample—such as by employing fluorescent detection or chemiluminescent detection. In employing one of these techniques, the cells are stained with a fluorescent or chemiluminescent dye to activate or tag cells of interest. The fluorescent or chemiluminescent dye stained cell is excited by light and the emission of the excitation is then detected by a photosensor (e.g., a charge coupled device (CCD) camera) or film emulsion that captures a digital image of the sample and allows further data analysis.
The performing of fluorescent detection, chemiluminescent detection, and/or colorimetric detection according to existing techniques—specifically with respect to the use of film emulsion, complementary metal-oxide-semiconductor (CMOS) imagers, and/or CCD cameras to capture images—presents some drawbacks and limitations. For example, film emulsion is the conventional detection medium for chemiluminescent detection, but is characterized by non-linear response and limited dynamic range requiring multiple exposures, thereby resulting in a time-consuming and expensive imaging procedure. As another example, as chemiluminescent signals generated are normally weak and time-varying, relatively fast exposure (e.g., on the order of a minute), low noise, and high light detection efficiency is required for accurate image capture when using CCDs. Thus, limitations of the CCDs regarding operation at a low frame rate (due to the inherent sequential read-out thereof) and low temperature (to achieve a reasonable noise level) present challenges in accurately capturing the chemiluminescent signals. Still further, CCDs require a high efficiency optical lens to focus the large blot to small CCD chips (˜1 cm2)—with the optical lens adding to the cost of the high-end CCDs, increasing the size and vertical space of the imaging device (due to the large working distance of the CCD camera), and also causing problems with regard to light collection efficiency (due to the large working distance). Yet still another drawback of image capture via CCD is that the capturing of images can take approximately 3-20 minutes—depending on the desired exposure.
Other more recent attempts to provide a system that captures a digital image of the blot include a C-digit system released by LICOR Biosciences that utilizes a linear scanner with sixteen linear sensors. The linear scanner combines short working distance (like film emulsion) to maximize light collection efficiency and multiple small low cost linear sensor arrays to meet the data acquisition time requirement, but the scan time to scan the large area is still around multiple minutes per pass (i.e., 6-12 minutes). Additionally, there is a concern that during the scanning time (on order of 10 minutes), the transient behavior of the chemiluminescence in the blot itself will be changing. As such—as the linear scan is happening—the intensity at the beginning of the scan will be higher than the intensity of at the end of the scan (bottom of the scan), therefore introducing an artificial gradient in the measurement.
Regardless of the exact technique employed to provide an image capture of cells in a sample, it is recognized that such image capture is performed separately from the microbial enumeration or colony counting. That is, presently, there is no single system that provides for image capture and microbial enumeration in a “multi-tasking” environment, where a single system can provide for the performing of both tasks in a time efficient and effective manner.
Therefore, it would be desirable to provide a system that provides for both image acquisition of microbial cells in a sample and the enumeration of such cells—that overcomes the aforementioned drawbacks of conventional imaging techniques and associated systems. It would also be desirable for such a system to provide improved performance in regards to sensitivity, dynamic range, exposure time, and quantum efficiency, over a large acquisition area, while eliminating costly high-efficiency imaging optics such as are used with existing CCD image sensors, so as to provide a system at a reduced cost and size.